Summary Nucleoid structuring proteins found within bacterial cells play an important role in compacting and organizing DNA, but they often silence the transcription of genes that they sequester. Molecular events leading to the displacement or relocation of nucleoid structuring proteins are central to bacterial physiology and virulence, but the mechanistic details remain obscure, despite being widespread among the enterics, pseudomonads and even some Gram positive bacterial pathogens. An improved understanding of these processes could lead to the discovery of novel drug targets and/or development of new antibacterials. The long term goal of this project is to fully understand mechanisms of transcriptional silencing and anti-silencing in the bacterial pathogen Shigella. Many genes on the Shigella virulence plasmid are silenced by the histone-like nucleoid structuring protein (H-NS) and derepressed by a protein antagonist called VirB. VirB is essential for virulence gene regulation in all Shigella species because virB mutants are avirulent. Based on the research progress made through previous R15 awards, this project will probe the mechanistic details of VirB-dependent transcriptional anti-silencing using three specific aims. The overarching hypothesis of this project is ?VirB binds to DNA sites that contain a run of consecutive guanine nucleotides, oligomerizes along the DNA causing a localized change in DNA supercoiling, which ultimately relieves H-NS mediated transcriptional silencing.? In Aim 1, a run of consecutive guanines will be evaluated for their role in the regulation of the icsP promoter and the promoters of other virulence genes. Consecutive guanines are commonly found upstream of VirB binding sites. Using site- directed mutagenesis, the importance of the individual guanines will be evaluated in VirB binding and VirB- dependent regulation at a model VirB-regulated promoter. Subsequently, the need for consecutive guanines located immediately upstream of 3 other VirB-binding sites will be tested. In Aim 2, VirB oligomerization along DNA in vivo will be investigated and it will be determined if this causes localized changes in DNA supercoiling. An innovative genetic tool will be used to determine if VirB oligomerizes along DNA in vivo. Using this tool, a VirB derivative, predicted to be defective in oligomerization, will be tested for its ability to oligomerize along DNA. If this protein is found to have a bona fide oligomerization defect, its ability to cause changes in DNA supercoiling will then be evaluated. In Aim 3, a VirB-independent approach will be used to manipulate DNA supercoiling of a promoter region that is transcriptionally silenced by H-NS. Using a VirB- independent approach, DNA supercoiling of a promoter region that is transcriptionally silenced by H-NS will be manipulated. If changes in DNA supercoiling are found to relieve H-NS-mediated repression, then similar changes mediated by VirB or other transcriptional anti-silencers could cause transcriptional anti-silencing at other bacterial promoters. All experiments have been carefully selected so they are suitable for students at the University of Nevada Las Vegas, in keeping with the R15 funding mechanism.